Recombination between nucleic acids is a well-established phenomenon in molecular biology. Genetic recombination that requires strong sequence homology between participating nucleic acid sequences to occur is generally referred to as homologous recombination. While most genetic knockout strategies employ homologous recombination to achieve a targeted knockout, in certain systems the occurrence of genetic recombination can impact genetic manipulations detrimentally. In particular, homologous recombination events can adversely impact construction and production of vectors, particularly viral vectors (e.g., adenovirus, retrovirus, adeno-associated virus, herpes virus, etc.), where it is often desirable to maintain highly homologous sequences (e.g., identical polypeptide sequences) within a single, stable viral vector free of homologous recombination during, e.g., passage and/or propagation of viral vector through one or more host cells and/or organisms.
In bacteria, homologous recombination begins with a step that involves a single-stranded end (Meselson and Radding 1975 Proc. Natl. Acad. Sci. USA, 80: 358-361). In eukaryotes, a mechanism of double-strand break (DSB) (Szostak et al. 1983 Cell 33: 25-35) has been suggested. DSB appears to occur in two principal mechanisms of homologous recombination: one conservative, within which all the nucleic acid sequences participating in the recombination event are present in the recombination products (ibid), the other nonconservative, during which certain sequences are lost. In mammalian somatic cells, the majority of homologous recombination events by DSB appears to take place according to a nonconservative process (Lin et al. 1990 Mol Cell Biol. 10: 103-12).
During the production of recombinant proteins, recombination events within an expression plasmid (intramolecular homologous recombination) can, for example, lead to the excision of the expression cassette from the transgene, resulting in a loss of expression. Recombination events can also produce excision of an expression cassette which has been stably integrated into the genome of a host producer cell, thereby inducing a loss of stability. Because of the potential for deleterious impact of homologous recombination events during vector production, particularly viral vector production, a need exists for effective strategies to reduce or eliminate the occurrence of homologous recombination events during synthesis of vectors, particularly viral vectors. Accordingly, there is also a need for vector compositions that possess reduced susceptibility to the occurrence of homologous recombination events, particularly viral vectors comprising, within a single vector, nucleic acid sequences that are designed to protect against the occurrence of homologous recombination events between those sequences encoding highly homologous polypeptides.